Overexpression of the Yes-associated protein (YAP), and TP53 family members ΔNp63 and p73, have been independently detected in subsets of head and neck squamous cell carcinomas (HNSCCs). YAP may serve as a nuclear cofactor with ΔNp63 and p73, but the functional role of YAP and their potential relationship in HNSCCs are unknown. In this study, we show that in a subset of HNSCC lines and tumors, YAP expression is increased but localized in the cytoplasm in association with increased AKT and YAP phosphorylation, and with decreased expression of ΔNp63 and p73. In another subset, YAP expression is decreased but detectable in the nucleus in association with lower AKT and YAP phosphorylation, and with increased ΔNp63 and p73 expression. Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP. ΔNp63 bound to the YAP promoter and suppressed its expression. Transfection of a YAP-serine-127-alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death. Conversely, YAP knockdown enhanced cell proliferation, survival, migration and cisplatin chemoresistance. Thus, YAP function as a tumor suppressor may alternatively be dysregulated by AKT phosphorylation at serine-127 and cytoplasmic sequestration, or by transcriptional repression by ΔNp63, in different subsets of HNSCC. AKT and/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.
We have previously detected increased expression of mRNA encoding the Yes-associated protein (YAP) by molecular profiling among genes differentially expressed in both the murine skin and a subset of human head and neck squamous cell carcinoma (HNSCC) lines (Dong et al., 1997, 2001b; Lee et al., 2007). Amplification of the chromosomal region that encodes YAP, 11q21–22, is frequently detected in human HNSCC lines and tumors (Carey et al., 1993; Snijders et al., 2005). We also noted differences in intensity associated with cytoplasmic or nuclear distribution of the YAP protein among different HNSCC tumor specimens by immunohistochemistry (Lee et al., 2007). These observations suggested that altered expression and cellular distribution of YAP could be important in the molecular pathogenesis of HNSCC.
The YAP protein consists of two isoforms, containing one or two conserved WW domains, which mediate binding to PPxY motif proteins, including the Src family kinase, Yes, for which it is named (Sudol, 1994; Chen and Sudol, 1995; Sudol et al., 1995). Other important cancer-related binding partners of YAP identified include isoforms of the tumor suppressor TP53 family transcription factors, p63α, p73α and p73β, but not TP53 itself (Strano et al., 2001). YAP was shown to serve as a cofactor for p73α-p300-mediated target gene transcription of the proapoptotic gene Bax, and p73-dependent apoptosis in response to DNA damage (Strano et al., 2001, 2005). YAP has been reported to function as a transcriptional coregulator of p73-mediated apoptosis in certain nonmalignant and cancer cells (Strano et al., 2001, 2005; Basu et al., 2003; Howell et al., 2004; Levy et al., 2007, 2008; Matallanas et al., 2007; Danovi et al., 2008; Oka et al., 2008; Yuan et al., 2008). Paradoxically, however, YAP has been implicated as an oncogene in other primary or immortalized cells (Baldwin et al., 2005; Snijders et al., 2005; Overholtzer et al., 2006; Zender et al., 2006; Zhao et al., 2007; Zhang et al., 2008).
The basis for differences in the function of YAP in these different contexts seems to be complex; potentially involving post-translational modification by phosphorylation through different signal proteins and interaction with different transcription factors (Downward and Basu, 2008; Bertini et al., 2009). Of potential relevance to HNSCC, phosphorylation of YAP serine-127 by AKT was reported to induce cytoplasmic sequestration by 14-3-3 and attenuation of p73 mediated apoptosis (Basu et al., 2003), and phosphoinositide 3-kinase (PI3K)-AKT activation is prevalent and implicated in the pathogenesis of HNSCC (Bancroft et al., 2002; Amornphimoltham et al., 2004, 2008; Massarelli et al., 2005; Molinolo et al., 2007; Bian et al., 2009; Moral et al., 2009). Furthermore, p63 and p73 with which YAP has been reported to interact (Strano et al., 2001) includes both proapoptotic and antiapoptotic isoforms (Barbieri and Pietenpol, 2006; Rosenbluth and Pietenpol, 2008), and overexpression of isoform ΔNp63α has been implicated in the dysregulation of p73 function, apoptosis and cell survival in a subset of HNSCCs (Rocco and Ellisen, 2006; Rocco et al., 2006). Thus, the role of AKT, ΔNp63, p73 and YAP in the dysregulation of apoptosis may be of particular significance in HNSCCs, in which the tumor-suppressor function of TP53 is frequently dysregulated by mutation or inactivation (Forastiere et al., 2001; Friedman et al., 2007).
We hypothesized that differences in YAP expression and cellular distribution could be related to alterations in AKT, ΔNp63 and p73, and could affect the function of YAP in HNSCCs.
Increased expression and cytoplasmic distribution of YAP is associated with increased YAP and AKT phosphorylation in HNSCC tissue array and cell lines
Based on the previous observations that YAP expression and/or cellular localization varies in murine and human HNSCCs (Dong et al., 2001b; Lee et al., 2007), we analyzed YAP expression and distribution patterns in a human tissue microarray with 20 HNSCC and 6 normal mucosa specimens. This revealed two major subsets of tumors: one subset showing high and predominantly cytoplasmic YAP staining (Figure 1a, higher resolution Supplementary Figure 1a, left panels) and the other showing relatively lower YAP staining with predominantly nuclear localization (Figure 1a, Supplementary Figure 1a, right panels). By a semi-quantitative histoscore, higher overall YAP staining intensity was observed in those tumors with a predominantly cytoplasmic pattern (Figure 1b, upper panel bar graphs). AKT is reported to be a potential modulator of Ser127 phosphorylation and cellular distribution of YAP (Basu et al., 2003), and exhibits increased phosphorylation in a subset of UMSCC lines (Bancroft et al., 2002) and HNSCC tumors (Amornphimoltham et al., 2004). To investigate whether increased YAP expression and cytoplasmic distribution is potentially related to YAP and AKT phosphorylation, we compared phospho-AKT (serine-473) and phospho-YAP (serine-127) in the same 20 HNSCC tissue microarray specimens. Increased cytoplasmic YAP in the tumor tissue was associated with higher phospho-AKT and phospho-YAP staining (Figure 1a, left panel; Figure 1b, lower panel bar graphs). Ordinary least squares analysis showed a significant relationship between increasing histoscores for phospho-AKTserine-473 and phospho-YAPserine-127 (Supplementary Figure 2a; r=0.5; P<0.05).
To determine whether HNSCC cell lines reflect and provide a model for the study of the differences observed in human tumors, we examined YAP mRNA and protein expression among a panel of nine UMSCC and a primary keratinocyte cell lines (Figure 1c). Compared with nonmalignant human keratinocytes (H), YAP mRNA and protein was increased in UMSCC lines 1–11B, and at relatively lower levels in UMSCC-22A, UMSCC-22B, UMSCC-38 and UMSCC-46 (Figure 1c; YAP mRNA expression; whole-cell extracts). Similar to HNSCC tumors, increased YAP expression was associated with both higher phospho-YAPserine-127 and phospho-AKTserine-473 levels. Total AKT and phospho-AKT was relatively lower in two lines with lower phospho-YAP, UMSCC-22A and UMSCC-22B. In addition, similar to HNSCC tumors, most of the UMSCC lines with high overall YAP expression, displayed higher cytoplasmic and lower nuclear YAP, whereas cells with low overall YAP mRNA and protein expression displayed relatively greater nuclear YAP (Figure 1c; cytoplasmic and nuclear extracts). Furthermore, for those with increased overall and cytoplasmic YAP, localization of phospho-YAPserine-127 was enriched in the cytoplasm (Figure 1d; cytoplasmic and nuclear extracts). The YAP cytoplasmic chaperone 14-3-3 did not differ between UMSCC cell lines (Supplementary Figure 2b, lower panels), excluding differences in 14-3-3 expression as a basis for the distribution observed. Analysis of HNSCC cell lines expressing higher (UMSCC-11A) and lower levels (UMSCC-22B) of YAP (Figure 1c) showed a similar pattern of YAP immunofluorescent staining intensity and cellular distribution as observed in the tumor tissue and subcellular extracts from cell lines (Supplementary Figure 3).
Pharmacological AKT inhibition or overexpression of a YAPserine-127 phosphoacceptor mutant enhances nuclear localization of YAP and cell death
To investigate the potential role of AKT activation in the phosphorylation and cellular distribution of YAP, the effect of inhibitor AKT-X previously shown to specifically inhibit both AKT phosphorylation and activation was examined (Thimmaiah et al., 2005). After exposure to 5 μM AKT-X for 1 h, AKTserine-473 and YAPserine-127 phosphorylation was inhibited (Figure 2a; left and center panels). The inhibition of phospho-AKT and phospho-YAP correlated with an increase in detection of YAP in the nucleus, with minimal change in the overall cytoplasmic levels of YAP (Figure 2a; center and right panels). To further investigate whether the limited nuclear YAP detected in cell lines with high YAP expression was due to serine-127 phosphorylation, we transfected UMSCC-11A cells with expression vectors encoding YAP2 (Y2) or YAP2-S127A mutant proteins (Y2M), as YAP2 with a second WW domain can be distinguished from endogenous YAP1 by its slower mobility (Figure 2b). An ∼50% increase in nuclear localization of the YAP protein was seen with mutant compared with the wild-type protein when normalized to OCT-1 (Figure 2b). We assessed the functional effect of increased nuclear S127A mutant YAP2 on the viability of UMSCC cells by flow cytometric DNA content analysis of sub-G0/G1-fragmented DNA (a measure of % cell death) 3 days after transfection with control vector, Y2 or Y2M (Figure 2c). The increase in the nuclear YAP2 mutant was accompanied by a relatively greater increase in cell death compared with empty control or wild-type YAP vectors, indicating that with phospho-site inactivation and increased nuclear localization, transfected YAP2 may function as a proapoptotic factor.
YAP and p53 family members ΔNp63 and p73 are differentially expressed in an inverse pattern in subsets of HNSCC cell lines, tumors and mucosal epithelia
The cell lines with higher YAP mRNA and protein expression in Figure 1c were previously found to be of wild-type TP53 genotype but weakly expressing TP53 protein (UMSCC-1, UMSCC-6, UMSCC-9 and UMSCC-11A), or a functionally deficient mutant (mt) TP53 protein (UMSCC-11B) (Friedman et al., 2007) (Figure 3a). In contrast, those with lower YAP expression in Figure 1c were found to overexpress mutant TP53 (Friedman et al., 2007) (Figure 3a). Examining the expression of other p53 family members in UMSCCs showed lower or undetectable expression of p63, p73 and wild-type TP53 together in one subset, whereas p63, p73 and mt TP53 were strongly expressed together in the other subset of UMSCC lines (Figure 3a). The identities of major p63 and p73 isoforms detected in UMSCC lines included ΔNp63α and TAp73, as determined by quantitative reverse transcriptase-PCR with specific primers; by comparison with electrophoretic mobilities of overexpressed plasmids containing ΔNp63α TAp63α and TAp63γ; and by blotting with TAp73-specific antibody (H Lu, unpublished data, not shown).
To examine whether the expression patterns of YAP, ΔNp63, p73 and/or TP53 detected in vitro are observed in HNSCC specimens in situ, we performed an immunohistochemistry staining of YAP, ΔNp63, p73 and TP53 in a panel of frozen human HNSCCs and squamous mucosal tissue specimens (Figure 3b). The strong and predominately cytoplasmic pattern of YAP staining was associated with relatively weak ΔNp63 and p73 staining in a subset of tumors (I–III). The relatively weak, but a predominantly nuclear YAP staining pattern was observed together with strong ΔNp63 and p73 staining in another subset (IV–VI). Although the inverse relationship between YAP and p63/p73 family members seen in UMSCC lines was observed in these tumor specimens, no clear association with TP53 immunostaining was observed in them. A similar pattern was observed in different hyperplastic nonmalignant mucosa (Figure 3b; right panels, compare HNSCC I–III and mucosa I; HNSCC IV–VI and mucosa II). Taken together, these data suggest that the expression and cytoplasmic distribution of YAP is often inversely associated with p63 and/or p73 in subsets of HNSCC and hyperplastic squamous mucosa.
ΔNp63 inhibits YAP expression, binds the YAP promoter and suppresses cell death
To examine whether the apparent inverse relationship between YAP and ΔNp63/p73 expression observed was potentially due to repression of YAP expression by ΔNp63 and/or p73, we explored the effects of small interfering RNA (siRNA) knockdown of p63 isoforms or p73 on YAP expression in UMSCC-11A, UMSCC-6 or UMSCC-22B. In pilot experiments, ⩾50% inhibition of targeted mRNA isoforms was observed after ΔNp63, TAp63, total p63 or p73 siRNA knockdown (Supplementary Figures 4a–c; upper panels). ΔNp63 knockdown resulted in a marked increase in YAP mRNA in UMSCC-11A, UMSCC-6 and UMSCC-22B at 48 h (Supplementary Figures 4a–c; lower panels). Knockdown of p73 had a relatively weaker but detectable effect in enhancing YAP expression (Supplementary Figures 4a and c). After ΔNp63 knockdown, both UMSCC-11A and UMSCC-6 lines displayed a greater increase in YAP expression when compared with the UMSCC-22B line (Supplementary Figure 4d).
Further experiments were conducted in UMSCC-11A, as this line expressing ΔNp63 at an intermediate level (Figure 3a) could be used for both knockdown and overexpression experiments with high transfection efficiencies. ΔNp63 knockdown resulted in a greater increase in YAP mRNA expression when compared with TAp63 knockdown during days 2 and 3 after treatment (Figure 4a). This was consistent with detection of only the ΔNp63 isoform protein by western blot in UMSCC lines by us (Figure 3a; H Lu, data not shown) and in other HNSCC lines by independent investigators (Rocco et al., 2006). ΔNp63 knockdown also had a greater effect on YAP protein expression at 3 days after treatment, and particularly in increasing the proportion of a faster migrating band that potentially represents a nonphosphorylated YAP capable of enhanced nuclear distribution (Figure 4b; middle lane). Based on the effect of ΔNp63 knockdown induced YAP expression, we hypothesized that ΔNp63 may serve as a negative regulator for YAP gene expression. To further investigate this possibility, we also overexpressed either ΔNp63 or TAp63 in UMSCC-11A. We observed a significantly greater decrease in YAP mRNA after expression of ΔNp63 than TAp63 (Figure 4c). Further support for direct transcriptional repression of YAP by ΔNp63 was obtained by demonstration of p63 binding to two regions of the YAP gene promoter containing predicted p63-binding sites by chromatin immunoprecipitaton assay (Figure 4d; Supplementary Figure 5). Thus, our data are consistent with a regulatory interaction underlying the inverse relationship between YAP and ΔNp63 expression observed in UMSCC cell lines and tumors; specifically, the knockdown or overexpression results established ΔNp63 as a repressor of YAP gene expression.
As ΔNp63 is the dominant isoform expressed by HNSCC, is able to repress YAP expression, and to hinder the proapoptotic function of p73 (Basu et al., 2003; Rocco et al., 2006), we compared the effects of p63 knockdown and of overexpression of YAP2S127A mutant vector on apoptosis. Knockdown of ΔNp63 or transient expression of mutant YAP vector both increased cell death by a similar increment (Figure 4e). No significant increase in cell death was observed by combining upregulation of YAP2S127A with ΔNp63 knockdown (Figure 4e). The knockdown of ΔNp63 resulted in an increase in annexin V and propidium iodide double-positive cells, consistent with cell death by apoptosis (Figure 4f). Knockdown of ΔNp63 over a longer term significantly decreased cell density in 5-day MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (XP Yang, data not shown).
YAP knockdown promotes cell proliferation, survival, migration and cisplatin chemotherapy resistance
To directly characterize the functional role of endogenous YAP, we inhibited YAP gene expression by siRNA in UMSCC-11A cells. Efficient inhibition of YAP mRNA was observed for up to 4 days, and knockdown of the YAP protein was also confirmed 3 days after transfection (Figure 5a). YAP inhibition was accompanied by effects on important genes implicated in the malignant phenotype of HNSCC (Figure 5b), including a significant decrease in mRNA expression of p53/p73 targets p53AIP1 and p21, which function to promote apoptosis and inhibit cell growth, as well as an increase in the expression of BCL-XL and VEGF, which promote cell survival and angiogenesis, respectively (Oda et al., 2000; Bancroft et al., 2002; Lee et al., 2008).
Upon YAP siRNA knockdown, a relative increase in proliferation was observed, indicating that YAP has residual antiproliferative and/or proapoptotic functional activity even in UMSCC-11A cells, which express relatively low levels of p73 and nuclear YAP (Figure 5c). The proapoptotic function of YAP was supported by a decrease in the percentage of sub-G0/G1-fragmented DNA (% cell death) and annexin V and propidium iodide double-positive cells (% apoptosis) (Figure 5d). An in vitro wound healing assay showed a significant increase in the rate of wound closure, also implicating YAP as a potential inhibitor of cell migration (Figure 5e). Inhibiting YAP expression also decreased the sensitivity of cells to the DNA-damaging agent cisplatin (Figure 5f). Taken together, these data indicate that YAP expressed in UMSCC cell lines inhibits cell proliferation, survival, migration and enhances cisplatin chemosensitivity.
In this study, we show that in HNSCC tissues, YAP is predominantly localized in the cytoplasm when overexpressed; whereas YAP is predominatly detected in the nucleus when underexpressed. An independent survey of YAP expression in four cancer types suggests that similar subsets may exist within other cancers of the same histological classification (Steinhardt et al., 2008). We provide evidence that in HNSCCs, these different patterns of YAP expression and cellular distribution are linked to increased AKT and YAP phosphorylation or ΔNp63 and p73 overexpression in subsets of tumors and cell lines (Figures 1 and 3). Where YAP expression is increased, it is predominantly detected in the cytoplasm in HNSCCs exhibiting increased AKT and YAP phosphorylation, but relatively low levels of ΔNp63 and p73. By contrast, YAP expression was markedly reduced but was detectable in the nucleus in another subset of tumors and lines with lower AKT and YAP phosphorylation, but increased ΔNp63 and p73. This previously unrecognized relationship suggested that alternative mechanisms may predominantly contribute to the regulation and function of YAP among these major subsets of HNSCC tumors and cell lines.
Cytoplasmic sequestration of YAP by 14-3-3 and inactivation as a cofactor for p73-mediated apoptosis was previously shown to involve phosphorylation of YAPser127, and YAP was reported to be a direct substrate of AKT in cells and by kinase assay of recombinant proteins in vitro (Basu et al., 2003). In HNSCC, we found a strong relationship between increased AKTser473 and YAPser127 phosphorylation, and cytoplasmic distribution in a subset of both tumors and cell lines (Figure 1). Furthermore, a selective AKT inhibitor, AKT-X, inhibited YAPser127 phosphorylation and enhanced nuclear YAP in HNSCC lines (Figure 2). Further supporting the hypothesis that AKT-dependent phosphorylation of YAPser127 contributes to the predominant sequestration of YAP in the cytoplasm and attenuation of apoptosis, expression of a YAP S127A phosphoacceptor mutant enhanced nuclear YAP and apoptosis. Consistent with these findings, we have shown that increased activation and phosphorylation of the PI3K and AKT signal axis in HNSCCs may occur as a result of overexpression and activation of the epidermal growth factor receptor, hepatocyte growth factor receptor (c-MET) or their ligands (Dong et al., 2001a; Bancroft et al., 2002). Furthermore, inhibitors of PI3K-AKT showed potent inhibition of tumor cell growth (Bancroft et al., 2002). Activation of the PI3K-AKT pathway has subsequently been strongly linked to development, cell survival and decreased prognosis of HNSCC (Amornphimoltham et al., 2004, 2008; Massarelli et al., 2005; Molinolo et al., 2007; Bian et al., 2009; Moral et al., 2009). However, a role for AKT in cytoplasmic inactivation of YAP as a contributing mechanism to cell survival in a subset of HNSCCs as described in this study has not been previously reported.
In the same subset in which YAP is most strongly expressed in the cytoplasm, decreased expression of members of the p53 family may also represent important codeterminants of dysfunction of YAP and apoptosis. The significant decrease in the expression of both TP53 and p73 (Figure 3) represents a further significant deficiency and compromise of the necessary components of the p73/p53 pathways critical for induction of apoptosis. YAP has previously been shown to be an important factor for preventing the degradation of p73 (Strano et al., 2005; Levy et al., 2007, 2008); hence, a study of how YAP cytoplasmic sequestration contributes to decreased p73 protein levels observed in this subset of UMSCC lines and HNSCC tumor specimens is warranted.
Protein–protein interactions between ΔNp63, p73 and/or YAP have been reported, and suggest that ΔNp63 can inhibit p73-YAP function (Strano et al., 2001; Rocco et al., 2006). We also examined the possibility that ΔNp63 and p73 overexpression potentially mediates negative feedback inhibition of expression of their cofactor. Supporting this hypothesis, knockdown of ΔNp63, and to a lesser extent, p73, enhanced YAP mRNA and protein expression, whereas overexpression of ΔNp63 repressed YAP mRNA expression. Analysis of the YAP promoter revealed p63-binding sites, and p63 was detected to bind to the regions of the YAP promoter containing these sequences, supporting ΔNp63 as a negative transcriptional regulator of YAP. Expression of other key growth arrest and apoptotic genes are inhibited by ΔNp63, including p21Cip1, NOXA and PUMA (Westfall et al., 2003; Rocco and Ellisen, 2006; Rocco et al., 2006), indicating that YAP may be one of several important tumor-suppressor genes repressed by overexpression of ΔNp63 in this HNSCC subset. These findings indicate that further analysis for common mechanisms of interaction of ΔNp63, p73 and YAP on the promoter of YAP and these other genes are warranted.
Although ΔNp63 is reported to repress expression of YAP and other proapoptotic genes, promoting survival of HNSCC (this study; Rocco et al., 2006), other reports suggest that p63 may repress other features of the malignant phenotype that affect the prognosis of SCC. p63 is principally immunolocalized in the basilar layer as shown in Figure 3b, where it has a role in regulating both regeneration and differentiation of the normal squamous epithelia (reviewed in the study by Barbieri and Pietenpol, 2006). p63 depletion by siRNA targeting total p63 was linked to modulation of multiple genes that enhanced malignant conversion, invasiveness and epithelial–mesenchymal transition of immortalized keratinocyte and HNSCC lines (Barbieri et al., 2006). Thus, the differential expression of p63 and phenotype of SCC in HNSCC subsets could reflect origination from p63-positive or p63-negative progenitors within the epithelia, or different stages of invasive SCC. However, we find that with specific knockdown of ΔNp63, migration is inhibited (XP Yang, unpublished data), consistent with the inhibitory effects of YAP (Figure 5e).
The role of YAP as a tumor suppressor in HNSCC was directly supported by the finding that increased YAP S127A mutant or endogenous YAP after ΔNp63 knockdown enhanced apoptosis, whereas YAP siRNA knockdown enhanced proliferation, survival, migration and resistance to the chemotherapeutic agent cisplatin. Consistent with these observations, overexpression of YAP has recently been implicated to enhance induction of apoptosis and detachment (Oka et al., 2008), whereas knockdown of YAP suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice (Yuan et al., 2008). With regard to chemotherapy sensitivity, YAP has been reported to be a critical component of DNA damage-induced cell death (Strano et al., 2001, 2005; Basu et al., 2003; Levy et al., 2007; Danovi et al., 2008; Oka et al., 2008). In addition, YAP has been reported to positively influence the expression of proapoptotic proteins BAX and PIG3 (Strano et al., 2001; Howell et al., 2004; Levy et al., 2008) and p53AIP1 (Oda et al., 2000), which have important roles in mediating either TP53- or p73-dependent apoptosis.
The role of YAP seems likely to be dependent on the molecular and tissue context, as there is now considerable evidence that YAP can serve a pro-oncogenic function in other nonmalignant and malignant cell types (reviewed in the study by Bertini et al., 2009). In these contexts, cytoplasmic homeostasis of YAP, and its drosophila ortholog Yorkie (Yki), was shown to be regulated by a conserved pathway that includes MST1/2/Hippo and LATS1/2/Wrts kinases, which when mutated or physiologically inactivated, result in reduced phosphorylation and nuclear translocation of YAP/Yki, and promotion of tissue overgrowth or tumorigenesis. Furthermore, several studies have provided evidence that LATS1/2, but not AKT, directly phosphorylates YAPser127. In addition, YAP is reported to associate with and undergo tyrosine phosphorylation by the YES/SRC family kinases, promoting interaction with transcription factor RUNX2 and osteoblastogenesis, or c-ABL, promoting interaction with p73, and apoptosis. Taken together, these observations suggest that YAP is a target and cofactor for several signal-activated transcription factors with opposing functions that are critical in cell survival. Hopefully, better understanding of the functions of YAP in different experimental systems will emerge from characterization of the net effects of upstream signaling that modulate YAP phosphorylation and localization, and alterations in TP53/ΔNp63/p73 that affect YAP expression or apoptosis, which together may determine YAP function.
In summary, we identified at least two subsets of HNSCCs with predominantly different mechanisms of dysregulation of YAP tumor-suppressor function (Figure 6). The mechanisms of dysregulation include AKT-dependent serine-127 phosphorylation and cytoplasmic sequestration, and a previously unreported mechanism, wherein overexpression of ΔNp63 represses YAP gene expression. Targeting YAP phosphorylation or ΔNp63 may enhance YAP nuclear expression, inhibit cell growth and migration, as well as enhance apoptosis and chemosensitization in HNSCCs.
Materials and methods
UMSCC cell lines (from Dr TE Carey; University of Michigan, Ann Arbor, MI, USA) were previously shown to reflect molecular and phenotypic alterations important in the pathogenesis of HNSCC (Chen et al., 1999; Dong et al., 2001a; Worden et al., 2005; Lee et al., 2006; Yu et al., 2006; Yan et al., 2007). Normal human epidermal keratinocytes (HEKA) were purchased from Invitrogen (Carlsbad, CA, USA). Cells were cultured and used as described previously (Chen et al., 1999; Yan et al., 2007).
Plasmids, siRNA and drug treatment
Human YAP expression vectors were constructed as described previously (Komuro et al., 2003). siRNAs specifically targeting YAP and TAp63 were from ON-TARGET plus of SMART pool selection (Dharmacon, Lafayette, CO, USA). The sequence of p63 siRNA targeting the unique exon in the N terminal of ΔNp63 was as published previously (Thurfjell et al., 2005) and was synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). The knockdown specificities and efficiencies were confirmed (XP Yang, unpublished data). Transfections were conducted with Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. Cisplatin (Bedford Laboratories, Bedford, OH, USA) was used as indicated. AKT inhibitor X (AKT-X, EMD Biosciences, Gibbstown, NJ, USA) used is 10-(4′-(N-diethylamino)butyl)-2-chlorophenoxazine, HCl; compound 10B (Thimmaiah et al., 2005).
Immunohistochemical staining of HNSCC tissue specimens
A tissue microarray with HNSCC and normal mucosa described in Supplementary Table 1 was stained for hematoxylin and eosin, pan-cytokeratin, YAP, p-YAP and p-AKT. Antibodies and methods used for immunostaining of the tissue microarray and frozen HNSCC specimens for YAP and p53 family members are described in Supplementary Methods. Immunohistochemistry histoscores based on the products of 0–3+ stain intensity multiplied by percentage positive cells per 100 in 3 different fields for each tumor section are described in Supplementary Methods.
Real-time quantitative reverse transcription–PCR
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation assays were performed using the EZ chromatin immunoprecipitation assay kit (Upstate Biotechnology, Waltham, MA, USA) according to the manufacturer's protocol, and as specified in Supplementary Methods.
MTT cell proliferation assay
Cells were trypsinized 24 h after siRNA transfection, transferred to a 96-well plate in quadruplicate, and were treated and/or analyzed 24 h after plating using an MTT Cell Proliferation kit (Roche Diagnostics, Indianapolis, IN, USA).
Cell cycle and apoptotic analysis by flow cytometry
Cells were processed using the Cycletest Plus DNA Regent Kit (BD Biosciences, San Jose, CA, USA) or the annexin V FITC (fluorescein isothiocyanate) and propidum iodide apoptotic Kit (Invitrogen) as per the manufacturer's instructions. All samples were run on a fluorescence-activated cell sorting Canto analyzer, and data were processed using Diva (BD Biosciences) or Flow-Jo (Tree Star Inc., Ashland, OR, USA).
Wound/scratch migration assay
Forty eight hours after siRNA transfections, wounds were made through confluent cell sheets. Measurements at preset distances on the wound were averaged and wound healing was quantified relative to the control siRNA.
Amornphimoltham P, Leelahavanichkul K, Molinolo A, Patel V, Gutkind JS . (2008). Inhibition of mammalian target of rapamycin by rapamycin causes the regression of carcinogen-induced skin tumor lesions. Clin Cancer Res 14: 8094–8101.
Amornphimoltham P, Sriuranpong V, Patel V, Benavides F, Conti CJ, Sauk J et al. (2004). Persistent activation of the Akt pathway in head and neck squamous cell carcinoma: a potential target for UCN-01. Clin Cancer Res 10: 4029–4037.
Baldwin C, Garnis C, Zhang L, Rosin MP, Lam WL . (2005). Multiple microalterations detected at high frequency in oral cancer. Cancer Res 65: 7561–7567.
Bancroft CC, Chen Z, Yeh J, Sunwoo JB, Yeh NT, Jackson S et al. (2002). Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines. Int J Cancer 99: 538–548.
Barbieri CE, Pietenpol JA . (2006). p63 and epithelial biology. Exp Cell Res 312: 695–706.
Barbieri CE, Tang LJ, Brown KA, Pietenpol JA . (2006). Loss of p63 leads to increased cell migration and up-regulation of genes involved in invasion and metastasis. Cancer Res 66: 7589–7597.
Basu S, Totty NF, Irwin MS, Sudol M, Downward J . (2003). Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis. Mol Cell 11: 11–23.
Bertini E, Oka T, Sudol M, Strano S, Blandino G . (2009). YAP: at the crossroad between transformation and tumor suppression. Cell Cycle 8: 49–57.
Bian Y, Terse A, Du J, Hall B, Molinolo A, Zhang P et al. (2009). Progressive tumor formation in mice with conditional deletion of TGF-beta signaling in head and neck epithelia is associated with activation of the PI3K/Akt pathway. Cancer Res 69: 5918–5926.
Carey TE, Van Dyke DL, Worsham MJ . (1993). Nonrandom chromosome aberrations and clonal populations in head and neck cancer. Anticancer Res 13: 2561–2567.
Chen HI, Sudol M . (1995). The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc Natl Acad Sci USA 92: 7819–7823.
Chen Z, Malhotra PS, Thomas GR, Ondrey FG, Duffey DC, Smith CW et al. (1999). Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer. Clin Cancer Res 5: 1369–1379.
Danovi SA, Rossi M, Gudmundsdottir K, Yuan M, Melino G, Basu S . (2008). Yes-associated protein (YAP) is a critical mediator of c-Jun-dependent apoptosis. Cell Death Differ 15: 217–219.
Dong G, Chen Z, Li ZY, Yeh NT, Bancroft CC, Van Waes C . (2001a). Hepatocyte growth factor/scatter factor-induced activation of MEK and PI3K signal pathways contributes to expression of proangiogenic cytokines interleukin-8 and vascular endothelial growth factor in head and neck squamous cell carcinoma. Cancer Res 61: 5911–5918.
Dong G, Loukinova E, Chen Z, Gangi L, Chanturita TI, Liu ET et al. (2001b). Molecular profiling of transformed and metastatic murine squamous carcinoma cells by differential display and cDNA microarray reveals altered expression of multiple genes related to growth, apoptosis, angiogenesis, and the NF-kappaB signal pathway. Cancer Res 61: 4797–4808.
Dong G, Loukinova E, Smith CW, Chen Z, Van Waes C . (1997). Genes differentially expressed with malignant transformation and metastatic tumor progression of murine squamous cell carcinoma. J Cell Biochem Suppl 28–29: 90–100.
Downward J, Basu S . (2008). YAP and p73: a complex affair. Mol Cell 32: 749–750.
Forastiere A, Koch W, Trotti A, Sidransky D . (2001). Head and neck cancer. N Engl J Med 345: 1890–1900.
Friedman J, Nottingham L, Duggal P, Pernas FG, Yan B, Yang XP et al. (2007). Deficient TP53 expression, function, and cisplatin sensitivity are restored by quinacrine in head and neck cancer. Clin Cancer Res 13: 6568–6578.
Howell M, Borchers C, Milgram SL . (2004). Heterogeneous nuclear ribonuclear protein U associates with YAP and regulates its co-activation of Bax transcription. J Biol Chem 279: 26300–26306.
Komuro A, Nagai M, Navin NE, Sudol M . (2003). WW domain-containing protein YAP associates with ErbB-4 and acts as a co-transcriptional activator for the carboxyl-terminal fragment of ErbB-4 that translocates to the nucleus. J Biol Chem 278: 33334–33341.
Lee TL, Yang XP, Yan B, Friedman J, Duggal P, Bagain L et al. (2007). A novel nuclear factor-kappaB gene signature is differentially expressed in head and neck squamous cell carcinomas in association with TP53 status. Clin Cancer Res 13: 5680–5691.
Lee TL, Yeh J, Friedman J, Yan B, Yang X, Yeh NT et al. (2008). A signal network involving coactivated NF-kappaB and STAT3 and altered p53 modulates BAX/BCL-XL expression and promotes cell survival of head and neck squamous cell carcinomas. Int J Cancer 122: 1987–1998.
Lee TL, Yeh J, Van Waes C, Chen Z . (2006). Epigenetic modification of SOCS-1 differentially regulates STAT3 activation in response to interleukin-6 receptor and epidermal growth factor receptor signaling through JAK and/or MEK in head and neck squamous cell carcinomas. Mol Cancer Ther 5: 8–19.
Levy D, Adamovich Y, Reuven N, Shaul Y . (2007). The Yes-associated protein 1 stabilizes p73 by preventing Itch-mediated ubiquitination of p73. Cell Death Differ 14: 743–751.
Levy D, Adamovich Y, Reuven N, Shaul Y . (2008). Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes in response to DNA damage. Mol Cell 29: 350–361.
Massarelli E, Liu DD, Lee JJ, El-Naggar AK, Lo Muzio L, Staibano S et al. (2005). Akt activation correlates with adverse outcome in tongue cancer. Cancer 104: 2430–2436.
Matallanas D, Romano D, Yee K, Meissl K, Kucerova L, Piazzolla D et al. (2007). RASSF1A elicits apoptosis through an MST2 pathway directing proapoptotic transcription by the p73 tumor suppressor protein. Mol Cell 27: 962–975.
Molinolo AA, Hewitt SM, Amornphimoltham P, Keelawat S, Rangdaeng S, Meneses Garcia A et al. (2007). Dissecting the Akt/mammalian target of rapamycin signaling network: emerging results from the head and neck cancer tissue array initiative. Clin Cancer Res 13: 4964–4973.
Moral M, Segrelles C, Lara MF, Martinez-Cruz AB, Lorz C, Santos M et al. (2009). Akt activation synergizes with Trp53 loss in oral epithelium to produce a novel mouse model for head and neck squamous cell carcinoma. Cancer Res 69: 1099–1108.
Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa C, Mori T et al. (2000). p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by ser-46-phosphorylated p53. Cell 102: 849–862.
Oka T, Mazack V, Sudol M . (2008). Mst2 and Lats kinases regulate apoptotic function of YAP. J Biol Chem 283: 27534–27546.
Overholtzer M, Zhang J, Smolen GA, Muir B, Li W, Sgroi DC et al. (2006). Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc Natl Acad Sci USA 103: 12405–12410.
Rocco JW, Ellisen LW . (2006). p63 and p73: life and death in squamous cell carcinoma. Cell Cycle 5: 936–940.
Rocco JW, Leong CO, Kuperwasser N, DeYoung MP, Ellisen LW . (2006). p63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 9: 45–56.
Rosenbluth JM, Pietenpol JA . (2008). The jury is in: p73 is a tumor suppressor after all. Genes Dev 22: 2591–2595.
Snijders AM, Schmidt BL, Fridlyand J, Dekker N, Pinkel D, Jordan RC et al. (2005). Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma. Oncogene 24: 4232–4242.
Steinhardt AA, Gayyed MF, Klein AP, Dong J, Maitra A, Pan D et al. (2008). Expression of Yes-associated protein in common solid tumors. Hum Pathol 39: 1582–1589.
Strano S, Monti O, Pediconi N, Baccarini A, Fontemaggi G, Lapi E et al. (2005). The transcriptional coactivator Yes-associated protein drives p73 gene-target specificity in response to DNA damage. Mol Cell 18: 447–459.
Strano S, Munarriz E, Rossi M, Castagnoli L, Shaul Y, Sacchi A et al. (2001). Physical interaction with Yes-associated protein enhances p73 transcriptional activity. J Biol Chem 276: 15164–15173.
Sudol M . (1994). Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product. Oncogene 9: 2145–2152.
Sudol M, Bork P, Einbond A, Kastury K, Druck T, Negrini M et al. (1995). Characterization of the mammalian YAP (Yes-associated protein) gene and its role in defining a novel protein module, the WW domain. J Biol Chem 270: 14733–14741.
Thimmaiah KN, Easton JB, Germain GS, Morton CL, Kamath S, Buolamwini JK et al. (2005). Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling. J Biol Chem 280: 31924–31935.
Thurfjell N, Coates PJ, Vojtesek B, Benham-Motlagh P, Eisold M, Nylander K . (2005). Endogenous p63 acts as a survival factor for tumour cells of SCCHN origin. Int J Mol Med 16: 1065–1070.
Westfall MD, Mays DJ, Sniezek JC, Pietenpol JA . (2003). The Delta Np63 alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay-Wells syndrome-derived mutations. Mol Cell Biol 23: 2264–2276.
Worden B, Yang XP, Lee TL, Bagain L, Yeh NT, Cohen JG et al. (2005). Hepatocyte growth factor/scatter factor differentially regulates expression of proangiogenic factors through Egr-1 in head and neck squamous cell carcinoma. Cancer Res 65: 7071–7080.
Yan B, Yang X, Lee TL, Friedman J, Tang J, Van Waes C et al. (2007). Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-kappaB and other signal transcription factors in head and neck squamous cell carcinoma. Genome Biol 8: R78.
Yu M, Yeh J, Van Waes C . (2006). Protein kinase casein kinase 2 mediates inhibitor-kappaB kinase and aberrant nuclear factor-kappaB activation by serum factor(s) in head and neck squamous carcinoma cells. Cancer Res 66: 6722–6731.
Yuan M, Tomlinson V, Lara R, Holliday D, Chelala C, Harada T et al. (2008). Yes-associated protein (YAP) functions as a tumor suppressor in breast. Cell Death Differ 15: 1752–1759.
Zender L, Spector MS, Xue W, Flemming P, Cordon-Cardo C, Silke J et al. (2006). Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell 125: 1253–1267.
Zhang J, Smolen GA, Haber DA . (2008). Negative regulation of YAP by LATS1 underscores evolutionary conservation of the Drosophila Hippo pathway. Cancer Res 68: 2789–2794.
Zhao B, Wei X, Li W, Udan RS, Yang Q, Kim J et al. (2007). Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev 21: 2747–2761.
We thank Drs Tsutomu Oka, Maie St John and Ms Cindy Clark for their critical review; Ning Yeh, Dr Liesl Nottingham, Dr Jay Friedman, Dr Yansong Bian and Jonah Cohen for technical assistance; and Dr Paul Albert for assistance with statistical analysis. This study was supported by the NIDCD Intramural project Z1ADC-000073, Z1ADC-000074, Head and Neck Cancer Charity Grant and the Breast Cancer Coalition Grant from the Pennsylvania Department of Health.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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Ehsanian, R., Brown, M., Lu, H. et al. YAP dysregulation by phosphorylation or ΔNp63-mediated gene repression promotes proliferation, survival and migration in head and neck cancer subsets. Oncogene 29, 6160–6171 (2010). https://doi.org/10.1038/onc.2010.339
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